The CD28-mediated costimulatory signal plays a pivotal role in the outcome of many immune responses including cytolytic responses in tumor and autoimmune diseases. Depending on the primary stimulation, CD28 can initiate multiple intracellular signaling pathways including signaling pathway insensitivity towards immunosuppressive drug, Cyclosporin A (CsA). This CsA-insensitive pathway is believed to be involved in graft-vs-host disease (GVHD) during allogeneic bone marrow transplantation. Our current objectives are to characterize the CsA-resistant costimulatory pathway and determine the physiological role of this signaling pathway in normal immune responses. Our initial studies have focused on the mechanism of activation of the IL-2 promoter in a CsA-resistant manner. Based on our preliminary data, we will focus our efforts on the role of protein phosphatase 2A (PP2A) instead of calcineurin in the activation of nuclear factor of activated T cells (NFAT) and Jun N-terminal kinase (JNK). Transient transfection in fresh peripheral blood lymphocyte will be used to examine the transcriptional activation of the IL-2 promoter through a CD28-responsive element. In addition, we will also investigate the role of PP2A in the CsA-resistant synergistic effects of IL-12 and CD28 on T cell activation. Furthermore, we propose to dissect the CsA-sensitive component of an allogeneic mixed lymphocyte reaction from the CsA-resistant component using different phosphatase inhibitors and neutralizing antisera (e.g., anti-IL-2, IL-12 and anti-IFN-m). We believe that further elucidation of this signaling pathway may assist in the identification of novel therapeutics to prevent GVHD.